Thrombolytic agents

ABSTRACT

The present invention relates to a thrombolytic agent capable of being orally administered, said agent comprising 6-amidino-2-naphthyl 4- (4,5-dihydro-1H-imidazole-2-yl)amino!benzoate or a pharmaceutically acceptable acid addition salt thereof.

DESCRIPTION

1. Technical Field

The present invention relates to a thrombolytic agent capable of beingorally administered, said agent comprising 6-amidino-2-naphthyl 4-(4,5-dihydro-1H-imidazole-2-yl)amino!benzoate or a pharmaceuticallyacceptable acid addition salt thereof.

2. Background Art

A clot formed by coagulation of blood in the heart or the blood vesselis called a thrombus, and a pathological process associated with theformation of said thrombus is called thrombosis. Thrombosis includes avariety of pathological states such as cerebral infarction, myocardialinfarction, pulmonary infarctionm etc.

The formation of thrombus is originally a mechanism that serves toprevent the potential leakage of blood from the injured part of theblood vessel when it is injured for some reason. The formation ofthrombus is closely associated with variation in blood components,abnormal blood circulation, and changes in properties of the walls ofblood vessels. That is, when a blood vessel is injured for some reason,blood platelets adhere to the injured site and aggregate to form anaggregated mass in order to prevent bleeding therefrom. Furthermore,platelets, by aggregating to one another, release substances thatactivate coagulation factors present in the blood to promote thrombusformation leading to the formation of more rigid thrombi. There are anumber of coagulation factors, which constitute the complex mechanism ofactivation in which the reaction proceeds in a cascade manner by oneenzyme activating the next (the blood coagulation system). Also, somecoagulation factors are directly activated by wounds or injured tissues,and the coagulation proceeds in a variety of ways. However, there are alot of substances present in the blood that suppress the progress ofcoagulation, thereby suppressing the enhancement of abnormalcoagulation. Blood clots are formed as needed but are lysed by theenzymes (the fibrinolytic enzymes) that dissolve clots in the blood whenthe hemostatic action becomes no longer needed and the blood vesselreturns to the original state (the fibrinolytic system).

Thus, thrombus formation involves a variety of factors. However, thefactors that are most directly involved in thrombus formation areplatelets, coagulation factors, fibrinolytic factors and the like, andconventionally drugs affecting the above mentioned blood components havebeen developed and used for treatment of thrombosis that are caused bythe presence of abnormal thrombi.

The methods of treatment of thrombosis are roughly divided into twogroups by the mechanism of action: the anti-thrombotic therapy thatprevents formation of thrombi, and the thrombolytic therapy thatdissolves the formed thrombi.

The anti-thrombotic therapy has further been classified into two: theanti-platelet therapy and the anti-coagulation therapy.

The anti-platelet therapy is intended to suppress the functions ofplatelets involved in the early stages of thrombus formation and drugssuch as classical aspirin and many other drugs that can be administeredorally have been developed. They are now used to prevent recurrence ofcerebral infarction, myocardial infarction, etc., to prevent occlusionafter various bypass surgeries, to prevent restenosis after coronaryangioplasty, and the like. Thus, they are used more as prophylacticdrugs against thrombus formation than as therapeutic drugs. They havethe problem of the presence of individual variation in appearance ofefficacy, and of bleeding tendency because of the need for prolongedadministration.

The anti-coagulation therapy is intended to inhibit thrombus formationby suppressing coagulation factors and is classified into the drugs forinhibiting activity of coagulation factors and the drugs for suppressingformation of coagulation factors. The former includes heparin, enzymeinhibitors, and the like. Since they are injections, they showanti-coagulation activity by intravenous administration. However, theyhave to be used under the supervision of a physician and also have theproblem of bleeding tendencies and the like. The latter is a method thatsuppresses the formation of coagulation factors by antagonizing vitaminK which is required for production of coagulation factors in vivo andthereby for inhibiting the overall coagulation ability. Therepresentative drug used is warfarin. Although warfarin can beadministered orally and has been used for chronic diseases that requireadministration for a prolonged period time, it has a problem of strongbleeding tendencies as a side effect.

The method that treats thrombosis in a mechanism of action differentfrom that of the anti-thrombotic therapy is the thrombolytic therapy.The purpose of the therapy is not suppression of the mechanism ofthrombus formation but recanalization of blood circulation by dissolvingthe thrombus in the blood vessel and thereby removing the occlusion ofthe blood vessel.

Its mechanism of action is believed to activate plasminogen, a precursorof the fibrinolytic system-modulating factors, into plasmin, whichplasmin dissolves thrombus by decomposing thrombus-forming fibrin in theblood vessel thus leading to recanalization of the occluded site. As thedrugs used in the thrombolytic therapy there are known endogeneoussubstances such as tissue plasminogen activator (t-PA), urokinase (UK),etc. that are plasminogen activating factors which activate plasminogeninto plasmin, microbially produced substances such as staphylokinase,streptokinase, etc., and recombinant formulation thereof. They are allinjections and no such drugs for oral administration are known.

It is assumed that t-PA etc. are generally useful in intravenousadministration. However, they have short half-lives and are quicklyeliminated from the liver. In addition, since there may be inhibitorspresent in vivo, an administration in a large dosage is required for thethrombolytic activity to function at the site of clots. It has beenreported that this transient administration of a large dose of athrombolytic drug remarkably enhances systemic activity of thrombolysis,and thus canalization of the occluded site is expected on the one hand,whereas the formed plasmin decomposes fibrinogen and other coagulationfactors, and injures the walls of the blood vessel thereby displayingbleeding symptoms on the other. Local administration of a thrombolyticdrug against coronary thrombosis at myocardial infarction is indeed avery effective method of treatment, but insertion of a catheter into theblood vessel is a special technique and poses a great burden to thepatient. Furthermore, it has been reported in the animal studies andclinical cases that although the administration of a thrombolytic agenttemporarily canalizes the occluded site, reocclusion may easily occur.

For the above-mentioned reasons, it has been desired to develop a drug:that has an activity of dissolving thrombus, a direct cause ofthrombosis, formed in the blood vessel; that does not show severe sideeffects such as bleeding, a problem associated with the conventionalthrombolytic agents, and the like, and; that can be orally administeredso that the burden on the patient may be alleviated.

DISCLOSURE OF THE INVENTION

As a result of intensive research in order to achieve the objectivementioned above, the present inventors have unexpectedly found that6-amidino-2-naphthyl 4- (4,5-dihydro-1H-imidazole-2-yl)amino!benzoatehas an excellent thrombolytic activity and we have completed the presentinvention. Thus, the present invention relates to a thrombolytic agentcapable of being orally administered, said agent comprising theabove-mentioned compound of the present invention or a pharmaceuticallyacceptable acid addition salt thereof. The compound of the presentinvention is a known compound that is useful as an anti-trypsin agent,anti-plasmin agent, anti-kallikrein agent, anti-thrombin agent andanti-complement agent as set forth in Japanese Unexamined PatentPublication No. 61('86)-33173. However, as is evident from thepharmacological studies mentioned below, the activity of the compound ofthe present invention to dissolve thrombus by activating thefibrinolytic system was discovered for the first time by the presentinventors, and the thrombolytic agent comprising the compound of thepresent invention and a pharmaceutically acceptable acid addition saltthereof is useful for diseases that are caused by thrombus. Thus, it canbe used as a drug for treatment of venous thrombosis, myocardialinfarction, pulmonary embolism, cerebral embolism, a slowly progressingcerebral thrombosis, and thrombotic embolism associated with vascularsurgery and extracorporeal blood circulation, as well as for improvementof circulation disorders, improvement of various conditions associatedwith chronic atrial occlusion, for treatment of thrombosis and embolismassociated with ischemic cerebrovascular disorders, and treatment ofthrombosis and embolism in general.

FIELD OF INDUSTRIAL APPLICATION

As is evident from the pharmacological studies below which shows thatthe compound of the present invention dissolves thrombus by activatingthe fibrinolytic system, the compound of the present invention can beused as a drug for treatment of venous thrombosis, myocardialinfarction, pulmonary embolism, cerebral embolism, a slowly progressingcerebral thrombosis, and thrombotic embolism associated with vascularsurgery and extracorporeal blood circulation, as well as for improvementof circulation disorders, improvement of various conditions associatedwith chronic atrial occlusion, for treatment of thrombosis and embolismassociated with ischemic cerebrovascular disorders, and treatment ofthrombosis and embolism in general.

Various pharmacological studies are illustrated below for the compoundof the present invention (6-amidino-2-naphthyl 4-(4,5-dihydro-1H-imidazole-2-yl)amino!benzoate mesilate).

(1) Improvement effects on mortality of mouse pulmonary thrombosislethal models

In the experiment, ddY male mice that had been fasted for six hours wereused. They were orally given 10 mg/kg of the compound of the presentinvention. Water was orally given to the control group. Six hours later,10 units/mouse of thrombin was intravenously given into the caudal veinto induce thrombosis. Alive or death of the animal was assessed 16 hoursafter thrombin induction. The effect of improvement on mortality by thecompound of the present invention was expressed in a survival rate. Theresults are shown Table 1.

                  TABLE 1    ______________________________________    Compound          Survival rate (%)    ______________________________________    the compound of the present                      66.7    invention    water              9.1    ______________________________________

(2) Determination of the plasmin-like activity in the plasma using asynthetic substrate

In the experiment, SD male rats that had been fasted overnight wereused. They were orally given 30 mg/kg of the compound of the presentinvention. Subsequently the blood was serially collected as the sodiumcitrate-added blood from the descending aorta and then the blood wascentrifuged to obtain plasma. Water was orally given to the controlgroup and plasma was obtained in a similar manner.

The plasma obtained was incubated with 0.1 M borate buffer, pH 8.5, and0.1 mM Boc-Val-Leu-Lys-MCA at 37° C. for 30 minutes. The reaction wasstopped by adding 15% acetic acid and then the intensity of fluorescencewas measured to determine the plasmin-like activity.

As a result, the group that was given the compound of the presentinvention has shown a marked enhancement in the plasmin-like activity ashigh as 0.968 nmol/min/ml as compared to 0.358 nmol/min/ml of thecontrol group.

(3) Determination of the tissue plasminogen activator (t-PA)-likeactivity using a synthetic substrate

The plasma obtained in the study example 2 mentioned above was incubatedwith 0.1 M borate buffer, pH 8.5, and 0.1 mM Phy-Gly-Arg-MCA at 37° C.for 30 minutes. The reaction was stopped by adding 15% acetic acid andthen the intensity of fluorescence was measured to determine thet-PA-like activity.

As a result, the group that was given the compound of the presentinvention has shown a marked enhancement in the t-PA-like activity ashigh as 1.351 nmol/min/ml as compared to 0.648 nmol/min/ml of thecontrol group.

(4) Determination of the amount of plasminogen activator inhibitor-1(PAI-1) antigen in the plasma.

In the experiment, SD male rats that had been fasted overnight wereused. They were orally given 30 mg/kg of the compound of the presentinvention. The blood was serially collected as the sodium citrate-addedblood from the descending aorta and then the blood was centrifuged toobtain plasma.

One hundred μl of a solution of monoclonal antibody to PAI-1 in 10 mMcarbonate buffer had been previously applied into a 96-well microtiterplate and the plate was left at 4° C. for 16 hours. After washing theplate four times with 10 mM phosphate buffer containing 0.1% Tween 20,the standard solution and the plasma samples were added. After leavingthe plate at room temperature for two hours, washing was repeated fourtimes again and 100 μl of the enzyme-labelled PAI-1 polyclonal antibodywas added and the plate was incubated at room temperature for two hours.After washing four times to remove excess antibody, 100 μl of thecitrate buffer containing 10 mg o-phenylenediamine and 0.1% hydrogenperoxide was added and was left at 30° C. for 30 minutes to develop thecolor. After stopping the reaction by adding 50 μl of 2 N sulfuric acid,absorbance at 405 nm was measured using the microplate reader todetermine the amount of PAI-1 antigen in the plasma samples. The resultis shown in FIG. 1.

In the control mechanism of thrombolysis, most of the t-PA released intothe blood is quickly inhibited by its inhibitor, PAI-1, to lose itsactivity and, therefore, the decrease in the amount of PAI-1 results inthe enhanced activity of thrombolysis. As shown in FIG. 1, the compoundof the present invention evidently caused a decrease in theconcentration of PAI-1, an inhibiting factor of fibrinolysis, in theblood.

(5) Determination of activated partial thromboplastin time (APTT)

To 100 μl of the reagent for determination of activated partialthromboplastin time (Platerin A-Auto) previously dissolved in distilledwater, 100 μl of the plasma sample obtained in the above study example 4was added, which was left at 37° C. for about three minutes. Then 100 μlof 0.25 M calcium chloride that had been prewarmed to 37° C. was addedand the time elapsed to the formation of fibrin clots was determined bythe coagulation meter (KC-10A, Amerung).

The result indicated no lengthening of APTT since the group that wasgiven the compound of the present invention coagulated at 20.3 secondsas compared to 20.2 seconds of the control group.

Subsequently, a study on oral administration to the human was conducted.

(6) Determination of human activated partial thromboplastin time (APTT)

The compound of the present invention was orally given to the normalhealthy individuals and blood was serially collected therefrom to obtainplasma.

To 100 μl of the reagent for determination of activated partialthromboplastin time (Platerin A-Auto) previously dissolved in distilledwater, 100 μl of the plasma sample obtained as above was added, whichwas left at 37° C. for about three minutes. Then 100 μl of 0.25 Mcalcium chloride that had been prewarmed to 37° C. was added and thetime elapsed to the formation of fibrin clots was determined by thecoagulation meter (KC-10A, Amerung). The result indicated no lengtheningof APTT since the group that was given the compound of the presentinvention coagulated at 18.4 seconds as compared to 18.5 seconds of thecontrol group.

(7) Determination of the amount of human PAI-1 antigen

When the amount of PAI-1 antigen was measured in a similar manner tothat in the study example 4 using the plasma obtained in the above studyexample 6, a marked decrease in the amount of PAI-1 antigen in theplasma was observed. The result is shown in FIG. 2.

(8) Determination of the amount of human PIC antigen

When the amount of plasmin-alpha₂ -plasmin inhibitor complex (PIC)antigen was also determined using the in-vitro diagnostic "PIC Test"(distributed by Teijin) for the plasma obtained in the above studyexample 6, a positive increase in the amount of PIC was observed. Theresult is shown in FIG. 3.

In thrombolysis, the fibrinolytic reaction that is mediated byplasminogen activator (PA) and plasmin plays an important role. α₂-plasmin inhibitor (α₂ -PI) is physiologically the most importantinhibiting factor of plasmin and serves as a regulating factor inthrombolysis. Since little PIC occurs in the plasma of the normalindividuals and is formed when plasmin is produced in vivo, theincreased amount of PIC indicates enhancement of the systemicfibrinolytic system.

Thus, the compound of the present invention, when orally given, exhibitsa very potent activity of thrombolysis.

Furthermore, in order to prove the effectiveness of the thrombolyticactivity of the compound of the present invention, the potency of saidcompound and an anticoagulant, heparin, were compared using anexperimental model in which thrombi had been previously formed in theblood vessel.

(9) A comparative test with the anticoagulant (heparin) in a ratthrombosis model

A comparative test on potency of the compound of the present inventionwith an anticoagulant, heparin, was conducted by constricting thecarotid arteries of the rat under anesthesia using the thrombosis modelthat produces thrombi at the constricted site (see Wessler Stanford, etal., J. Appl. Physiol. 14(6): 943-946 (1959)). This experimental modelis the one used to demonstrate the effectiveness of t-PA and UK thathave been used as thrombolytic agents.

In the experiment, SD male rats were used. The carotid veins of the ratswere exposed under anesthesia and then ligated together with a wire of 2mm in diameter. Five hundred units/ml of thrombin was injected into theblood vessel to produce a thrombin thrombus in the ligated vessel. Fiveminutes after the formation of the thrombus, the wire was removed andthe open vessels were sutured. After the righting reflex was restored,100 mg/kg of the compound of the present invention was orally given. Theanticoagulant heparin was intravenously administered. Three hours afterthe administration of the drugs, the rats were sacrificed and thecarotid vein that produced thrombus was extirpated. The residual thrombiin the extirpated blood vessel were counted based on the followingscoring to evaluate the effectiveness of the drugs. The results areshown in Table 2.

Criterion:

Score 0 No thrombus

Score 1 Microthrombus

Score 2 Several small thrombotic masses

Score 3 Several large thrombotic masses

Score 4 Thrombotic masses that turned into cruors

                  TABLE 2    ______________________________________    Compound             Score    ______________________________________    Compound of the present invention                         1    Water                4    Heparin              4    ______________________________________

From the above result, it was demonstrated that the compound of thepresent invention is very effective against those thrombi on whichanticoagulants have no effects.

It was also confirmed that the compound of the present invention, whenorally given to the healthy individuals, does not induce anyabnormalities in the subjective symptoms, the objective symptoms, orlaboratory test results, and that it does not have any safety problems.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows changes in the amount of PAI-1 antigen in the blood of therat after oral administration of the compound of the present invention.The horizontal axis denotes time (hours) after the administration, andthe vertical axis denotes the ratio of the PAI-1 concentration in theblood relative to the value set at 1.0 of the concentration of PAI-1 inthe blood immediately after the administration.

FIG. 2 shows changes in the amount of PAI-1 antigen in the blood of thehuman after oral administration of the compound of the presentinvention. The horizontal axis denotes time (hours) after theadministration, and the vertical axis denotes the amount (ng/ml) ofPAI-1 antigen in the blood.

FIG. 3 shows changes in the amount of PIC antigen in the blood of thehuman after oral administration of the compound of the presentinvention. The horizontal axis denotes time (hours) after theadministration, and the vertical axis denotes the amount (ng/ml) of PICantigen in the blood.

The compound of the present invention may be generally administered inthe form of pharmaceutical compositions. Examples of the abovecompositions include tablets, powders, capsules, syrups, or aqueoussolutions. For compositions intended for oral administration, commonlyused additives may be used such as excipients, lubricants,disintegrants, wetting agents, etc. The liquids intended for oraladministration may be in the from of aqueous solutions, oleaginoussolutions, solutions, emulsions, syrups, elixirs, etc., or they may bedispensed as dry syrups that are reconstituted with water or any othersuitable solvent prior to use. The above-mentioned liquids may containthe common additives such as a suspending agent, a flavoring agent, adiluent, or an emulsifying agent. It may also be used as a suppositoryfor rectal administration. The suppository may employ as the base anysuitable materials such as cacao butter, lauric butter, macrogol,glycerogelatin, Witepsol, sodium stearate, or mixtures thereof, and,when desired, an emulsifying agent, a suspending agent, a preservative,and the like may be added.

BEST MODE FOR CARRYING OUT THE INVENTION

The compound of the present invention may be administered to mammals(including human patients) in the form of drugs for oral administrationor for rectal administration. For the purpose of treating variousdiseases in the human caused by thrombus, the dosage is in the range of100 to 1000 mg/day/person, preferably 200 to 800 mg/day/person, and morepreferably 400 to 700 mg/day/person, but it can be adjusted asappropriate depending upon the severity of the disease, age and weightof the patient, etc.

We claim:
 1. A method for producing thrombolysis in an animalcomprising:administering to an animal in need thereof, an effectiveamount of 6-amidino-2-napthyl 4-(4,5-dihydro-1H-imidazole-2-yl)amino!-benzoate or a pharmaceuticallysuitable, salt thereof sufficient to produce thrombolysis.
 2. A methodfor producing thrombolysis in an animal comprising:administering to ananimal in need thereof, an effective amount of a composition comprising6-amidino-2-napthyl 4- (4,5-dihydro-1H-imidazole-2-yl)amino!-benzoate ora pharmaceutically suitable salt thereof and a pharmaceuticallyacceptable carrier sufficient to produce thrombolysis.
 3. A method forthe treatment of myocardial infarction comprising:administering to ananimal in need thereof, an effective amount of 6-amidino-2-napthyl 4-(4,5-dihydro-1H-imidazole-2-yl)amino!-benzoate or a pharmaceuticallysuitable salt thereof sufficient to produce thrombolysis.
 4. A methodfor the treatment of myocardial infarction comprising:administering toan animal in need thereof, an effective amount of a compositioncomprising 6-amidino-2-napthyl 4-(4,5-dihydro-1H-imidazole-2-yl)amino!-benzoate or a pharmaceuticallysuitable salt thereof and a pharmaceutically acceptable carriersufficient to produce thrombolysis.